powerpoint presentation materials to accompany genetics: analysis and principles robert j. brooker...

PowerPoint Presentation Materialsto accompany

Genetics: Analysis and PrinciplesRobert J. Brooker

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 10

CHROMOSOME ORGANIZATION AND MOLECULAR STRUCTURE

INTRODUCTION

Chromosomes are the structures that contain the genetic material complexes of DNA and proteins

The genome comprises all the genetic material that an organism possesses In bacteria, it is typically a single circular chromosome In eukaryotes, it refers to one complete set of nuclear

chromosomes Note:

Eukaryotes possess a mitochondrial genome Plants also have a chloroplast genome

10-2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

INTRODUCTION

The main function of the genetic material is to store information required to produce an organism The DNA molecule does that through its base sequence

DNA sequences are necessary for 1. Synthesis of RNA and cellular proteins 2. Proper segregation of chromosomes 3. Replication of chromosomes 4. Compaction of chromosomes

So they can fit within living cells


Viruses are small infectious particles containing nucleic acid surrounded by a capsid of proteins Refer to Figure 10.1

For replication, viruses rely on their host cells ie., the cells they infect

Most viruses exhibit a limited host range They typically infect only specific types of cells of one

host species


10.1 VIRAL GENOMES

10-4

Figure 10.1 General structure of viruses10-5

Bacteriophages may also contain a sheath, base plate and tail fibers

Refer to Figure 9.4Lipid bilayer

Picked up when virus leaves host cell

The genome can be DNA or RNA Single-stranded or double-stranded Circular or linear

Viral genomes vary in size from a few thousand to more than a hundred thousand nucleotides


Viral Genomes

10-6

During an infection process, mature viral particles need to be assembled


Viruses with a simple structure may self-assemble Genetic material

and capsid proteins spontaneously bind to each other

Example: Tobacco mosaic virus Figure 10.2

Capsid protein

Capsid composed of 2,130 identical protein subunits

The bacterial chromosome is found in a region called the nucleoid Refer to Figure 10.3

The nucleoid is not membrane-bounded


10.2 BACTERIAL CHROMOSOMES

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Fig. 10.3

Bacterial chromosomal DNA is usually a circular molecule that is a few million nucleotides in length Escherichia coli ~ 4.6 million base pairs Haemophilus influenzae ~ 1.8 million base pairs

A typical bacterial chromosome contains a few thousand different genes Structural gene sequences (encoding proteins) account

for the majority of bacterial DNA The nontranscribed DNA between adjacent genes are

termed intergenic regions

Figure 10.4 summarizes the key features of bacterial chromosomes



Figure 10.4

A few hundred nucleotides in length

These play roles in DNA folding, DNA replication, and gene expression

To fit within the bacterial cell, the chromosomal DNA must be compacted about a 1000-fold This involves the formation of loop domains


Figure 10.5

The number of loops varies according to the size of the bacterial chromosome and the species

E. coli has 50-100 with 40,000 to 80,000 bp of DNA in each

The looped structure compacts the chromosome about 10-fold

DNA supercoiling is a second important way to compact the bacterial chromosome


Figure 10.6

Figure 10.7 provides a schematic illustration of DNA supercoiling

Supercoiling within loops creates a more compact DNA

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Figure 10.7Plates preventing DNA

ends from rotating freely

Fewer turnsMore turns

Both overwinding and underwinding can

induce supercoiling

These three DNA conformations are topoisomers of each other

These two DNA conformations do not occur in living cells

The chromosomal DNA in bacteria is negatively supercoiled In E. coli, there is one negative supercoil per 40 turns of

the double helix

Negative supercoiling has two major effects 1. Helps in the compaction of the chromosome

Refer to Figure 10.6 2. Creates tension that may be released by DNA

strand separation Refer to Figure 10.8


Chromosome Function Is Influenced by DNA Supercoiling

10-16

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 10-17Figure 10.8

This enhances DNA replication and transcription

The control of supercoiling in bacteria is accomplished by two main enzymes

1. DNA gyrase (also termed DNA topoisomerase II) Introduces negative supercoils using energy from ATP

Refer to Figure 10.9 It can also relax positive supercoils when they occur

2. DNA topoisomerase I Relaxes negative supercoils

The competing action of these two enzymes governs the overall supercoiling of bacterial DNA


Eukaryotic species contain one or more sets of chromosomes

Each set is composed of several different linear chromosomes

The total amount of DNA in eukaryotic species is typically greater than that in bacterial cells

Chromosomes in eukaryotes are located in the nucleus To fit in there, they must be highly compacted

This is accomplished by the binding of many proteins The DNA-protein complex is termed chromatin


10.3 EUKARYOTIC CHROMOSOMES

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Eukaryotic genomes vary substantially in size Refer to Figure 10.10a

In many cases, this variation is not related to complexity of the species For example, there is a two fold difference in the size of

the genome in two closely related salamander species Refer to Figure 10.10b

The difference in the size of the genome is not because of extra genes

Rather, the accumulation of repetitive DNA sequences These do not encode proteins


Fig. 10.10

A eukaryotic chromosome contains a long, linear DNA molecule Refer to Figure 10.11

Three types of DNA sequences are required for chromosomal replication and segregation Origins of replication Centromeres Telomeres


Organization of Eukaryotic Chromosomes

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10-25Figure 10.11

Genes are located between the centromeric and telomeric regions along the entire chromosome A single chromosome usually has a few hundred to

several thousand genes

In lower eukaryotes (such as yeast) Genes are relatively small

They contain primarily the sequences encoding the polypeptides ie: Very few introns are present

In higher eukaryotes (such as mammals) Genes are long

They tend to have many introns


Sequence complexity refers to the number of times a particular base sequence appears in the genome

There are three main types of repetitive sequences Unique or non-repetitive Moderately repetitive Highly repetitive


Repetitive Sequences

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Unique or non-repetitive sequences Found once or a few times in the genome structural genes (control regions, exons and introns) intergenic areas

Moderately repetitive Found a few hundred to a few thousand times Includes

Genes for rRNA and histones Origins of replication Transposable elements

Discussed in detail in Chapter 17



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Highly repetitive Found tens of thousands to millions of times Each copy is relatively short (a few nucleotides to several

hundred in length)

Some sequences are interspersed throughout the genome Example: Alu family in humans

Discussed in detail in Chapter 17

Other sequences are clustered together in tandem arrays Example: AATAT and AATATAT sequences in Drosophila These are commonly found in the centromeric regions



10-29


Figure 10.13

Effect of base composition on DNA melting temperature

• Hyperchromic shift during DNA denaturation is used to determine the melting temperature (Tm) (Figure 10.20). Melting

temperature is a method for estimating the base composition of DNA.

•G:C base pairs more stable than A:T base pairs


If stretched end to end, a single set of human chromosomes will be over 1 meter long! Yet the cell’s nucleus is only 2 to 4 m in diameter

The compaction of linear DNA involves interactions between DNA and various proteins

Eukaryotic Chromatin Compaction

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The repeating structural unit within eukaryotic chromatin is the nucleosome

It is composed of double-stranded DNA wrapped around an octamer of histone proteins An octamer is composed two copies each of four different

histones 146 bp of DNA make 1.65 negative superhelical turns

around the octamer

Refer to Figure 10.14a

Nucleosomes

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Overall structure of connected nucleosomes resembles “beads on a string” This structure shortens the DNA length about seven-fold

Figure 10.14

Vary in length between 20 to 100 bp, depending on species and cell type

Diameter of the nucleosome


Histone proteins are basic They contain many positively-charged amino acids

Lysine and arginine These bind with the phosphates along the DNA backbone

There are five types of histones H2A, H2B, H3 and H4 are the core histones

Two of each make up the octamer

H1 is the linker histone Binds to linker DNA Also binds to nucleosomes

But not as tightly as are the core histones

Refer to Figure 10.14b

10-46


Figure 10.14

Play a role in the organization and compaction

of the chromosome

The model of nucleosome structure was proposed in 1974 by Roger Kornberg

Kornberg based his proposal on various observations about chromatin Biochemical experiments X-ray diffraction studies Electron microscopy images


Experiment 10B: Nucleosome Structure Revealed

10-48

Markus Noll tests Kornberg’s model

He did this via the following procedure Digest DNA with the enzyme DNase I Accurately measure the molecular weight of the

resulting DNA fragments

The rationale is that the linker DNA is more accessible than the “core DNA” to the DNase I


Experiment 10B: Nucleosome Structure Revealed

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The Hypothesis The experiment tests the beads-on-a-string model

of chromatin structure It the model is correct, DNase I should cut in the linker

region Thereby producing DNA pieces that are about 200 bp long


Testing the Hypothesis

Refer to Figure 10.15

10-50

10-51Figure 10.15

The Data


30 unitsml-1

Interpreting the Data


30 unitsml-1

All chromosomal DNA digested into fragments that are ~ 200 bp in length

These longer pieces were all in multiples of 200 bp

At low concentrations, DNase I did not cut at all

the linker DNA

This fragment contains two nucleosomes

And this, three


Nucleosomes associate with each other to form a more compact structure termed the 30 nm fiber

Histone H1 plays a role in this compaction At moderate salt concentrations, H1 is removed

The result is the classic beads-on-a-string morphology At low salt concentrations, H1 remains bound

Beads associate together into a more compact morphology


Nucleosomes Join to Form a 30 nm Fiber

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Fig. 10.16


The 30 nm fiber shortens the total length of DNA another seven-fold

Its structure has proven difficult to determine The DNA conformation may be substantially altered

when extracted from living cells

Two models have been proposed Solenoid model Three-dimensional zigzag model

Refer to Figure 10.17b and c

10-55


Figure 10.17

Regular, spiral configuration containing six

nucleosomes per turn

Irregular configuration where nucleosomes have little face-to-face

contact


The two events we have discussed so far have shortened the DNA about 50-fold

A third level of compaction involves interaction between the 30 nm fiber and the nuclear matrix

The nuclear matrix is composed of two parts Nuclear lamina Internal matrix proteins

10 nm fiber and associated proteins

Further Compaction of the Chromosome

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Figure 10.18


Figure 10.18

The third mechanism of DNA compaction involves the formation of radial loop domains

Matrix-attachment regions

Scaffold-attachment regions (SARs)

or

MARs are anchored to the nuclear

matrix, thus creating radial loops

25,000 to 200,000 bp


The attachment of radial loops to the nuclear matrix is important in two ways 1. It plays a role in gene regulation

Discussed in Chapter 15

2. It serves to organize the chromosomes within the nucleus

Each chromosome in the nucleus is located in a discrete and nonoverlapping chromosome territory


Further Compaction of the Chromosome

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Fig. 10.19

Euchromatin Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains

Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further

Heterochromatin vs Euchromatin

10-61

10-63Figure 10.21

10-64Figure 10.21

Compaction level in euchromatin

Compaction level in heterochromatin

During interphase most chromosomal

regions are euchromatic

Fig. 10.21cd


As cells enter M phase, the level of compaction changes dramatically By the end of prophase, sister chromatids are entirely

heterochromatic Two parallel chromatids have an overall diameter of

1,400 nm

These highly condensed metaphase chromosomes undergo little gene transcription

Metaphase Chromosomes

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Metaphase Chromosomes

Fig. 10.22a

Fig. 10.22b

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